Journal: Stem Cell Reports

Article Title: NOTCH-mediated ex vivo expansion of human hematopoietic stem and progenitor cells by culture under hypoxia

doi: 10.1016/j.stemcr.2021.08.001

Figure Lengend Snippet: NOTCH and hypoxia pathways cooperate in human HSPCs (A) Heatmap of leading edge subset (left) and enrichment plot (right) showing relative expression of genes associated with the NOTCH pathway in CD34 + cells cultured in normoxia (21% O 2 ) or hypoxia (1%–2% O 2 ) with optimized densities of DXI. NES, normalized enrichment score; FDR, false discovery rate. (B) Top: western blot analysis confirming exogenous HIF-1α expression in human CD34 + cells cultured under normoxic conditions with DXI after transduction with a mutant HIF-1α-expressing lentiviral vector. A control group transduced with a GFP-expressing lentiviral vector is shown for comparison. β-Tubulin was used as loading control. Bottom: HES-1 gene expression measured by qRT-PCR (n = 9 technical replicates, from three donors). Results indicate the fold increase in HES-1 levels normalized to a GFP-transduced control group. (C) Representative ImageStream images of human CD34 + cells cultured under normoxic or hypoxic conditions in the absence or with optimized concentrations of DXI. BF, bright-field; NICD, NOTCH1 intracellular domain (ICD); DAPI, 4′,6-diamidino-2-phenylindole. (D) Quantification of the mean fluorescence intensity (MFI) of NOTCH1 ICD in 10,000 cells/condition. (E) Representative high-resolution images of individual cells assessed by confocal microscopy. The bottom panel displays a colocalization channel generated with Imaris imaging software. (F) Quantification of NOTCH1 ICD and HIF-1α signal colocalization in individual cells for each condition by the Pearson's correlation coefficient (n = 20 cells/condition). In (B), (D), and (F), data are displayed as mean ± SEM. Two-sided (B and F) and one-sided (D) unpaired t test were used. ∗ p ≤ 0.05, ∗∗ p ≤ 0.01, ∗∗∗∗ p ≤ 0.0001. See also and ; .

Article Snippet: The bottom panel displays a colocalization channel generated with Imaris imaging software. (F) Quantification of NOTCH1 ICD and HIF-1α signal colocalization in individual cells for each condition by the Pearson's correlation coefficient (n = 20 cells/condition).

Techniques: Expressing, Cell Culture, Western Blot, Transduction, Mutagenesis, Plasmid Preparation, Control, Comparison, Gene Expression, Quantitative RT-PCR, Fluorescence, Confocal Microscopy, Generated, Imaging, Software

Journal: Circulation Research

Article Title: Notch Pathway Targets Proangiogenic Regulator Sox17 to Restrict Angiogenesis

doi: 10.1161/circresaha.115.303142

Figure Lengend Snippet: Figure 4. Sox17 expression is inversely regulated by Notch signaling. A, Immunofluorescence images for Sox17 (red) and green fluorescent protein (GFP; green) of human umbilical vein endothelial cells transfected with mock (control) or Notch1 intracellular domain (N1ICD) vector, which can express GPF simultaneously by a dual promoter system. Transfected cells are distinguished as GFP expression. Arrows highlight reduced Sox17 protein expression only in individual cells transfected with N1ICD vector but not in nontransfected cells. (B) Quantification of Sox17 protein expression in A. Images for Sox17 (red) and isolectin-B4 (IB4; green) of P5 retina. (C) Reduced Sox17 expression in N1ICDiGOF mutants. D, Increased Sox17- expressing endothelial cells on α-delta–like ligand 4 (Dll4) antibody treatment compared with IgG treatment. Scale bars, 100 μm. Ctrl indicates control.

Article Snippet: In Vitro Modulation of Notch Signaling For in vitro activation of Notch signaling, human umbilical vein endothelial cells were transfected with EF.hICN1.CMV.GFP vector (17623, Addgene) containing human Notch1 intracellular domain or EF.CMV.RFP vector (17619, Addgene) as a negative control.

Techniques: Expressing, Immunofluorescence, Transfection, Control, Plasmid Preparation

Journal: Circulation Research

Article Title: Notch Pathway Targets Proangiogenic Regulator Sox17 to Restrict Angiogenesis

doi: 10.1161/circresaha.115.303142

Figure Lengend Snippet: Figure 6. Notch pathway regulates Sox17 expression mainly at the post-transcriptional level. Immunoblotting and quantification of Sox17 signal (A, C, E, G, I) and transcript level of Sox17 (B, D, F, H, J). A to D, Reduced Sox17 protein level on Notch activation. A and B, Human umbilical vein endothelial cells (HUVECs) transfected with control or Notch1 intracellular domain (N1ICD) vector (n=4). C and D, P5 lungs from control and N1ICDiGOF pups (n=3). E to J, Increased Sox17 protein level after Notch inactivation. E and F, HUVECs treated with vehicle (control) or N-[N-(3,5-difluorophenacetyl)-L-alanyl]–S-phenylglycine t-butylester (DAPT; n=4). G and H, P5 lungs treated with IgG or α-delta-like ligand 4 (Dll4) antibody (Ab; n≥3). I, Images showing Sox17 (red) and platelet endothelial cell adhesion molecule (PECAM; blue) of Lewis lung carcinoma tumors treated with IgG or α-Dll4 Ab. Quantification of Sox17 intensity in tumor vessels (n=20; right). J, Transcript level of Sox17 in tumor endothelial cells (tECs) of tumors treated with IgG or α-Dll4 Ab (n=3). Data are presented as mean±SD; #P<0.01; *P<0.05. Scale bars, 100 μm.

Article Snippet: In Vitro Modulation of Notch Signaling For in vitro activation of Notch signaling, human umbilical vein endothelial cells were transfected with EF.hICN1.CMV.GFP vector (17623, Addgene) containing human Notch1 intracellular domain or EF.CMV.RFP vector (17619, Addgene) as a negative control.

Techniques: Expressing, Western Blot, Activation Assay, Transfection, Control, Plasmid Preparation

Journal: Journal of Cellular and Molecular Medicine

Article Title: Nitric oxide facilitates the S‐nitrosylation and deubiquitination of Notch1 protein to maintain cancer stem cells in human NSCLC

doi: 10.1111/jcmm.70203

Figure Lengend Snippet: NO sustains CSC stemness through the activation of Notch1. (A) Immunoblot analysis of Notch1 and Hes1 protein in non‐CSCs and CSCs. (B) The protein expression of Notch1 was analysed in non‐CSCs treated with DETA NONOate (20 μM) for 24 h. (C) CSCs were treated with L‐NAME for 24 h and detected for Notch1 protein with immunoblots. (D, E) CSCs were treated with L‐NAME (100 μM)/1400 W (100 μM) in the presence or absence of the DETA NONOate (20 μM)/GSNO (200 μM) for 24 h. Protein expression of Notch1 and Hes1 were detected by immunoblot. (F–H) CSCs were exposed to L‐NAME (100 μM) with or without the reintroduction of NICD and were analysed for expression of Notch1 protein, stem‐related transcripts and spheres formation. Mean ± SEM from 5 to 9 independent experiments. * p < 0.05, ** p < 0.01 and *** p < 0.001 with paired t ‐test (B, C), unpaired t ‐test (A) and ANOVA plus Turkey's method (D, G, H).

Article Snippet: Human Notch1 intracellular domain plasmid (#130934) was from Addgene.

Techniques: Activation Assay, Western Blot, Expressing

Journal: Journal of Cellular and Molecular Medicine

Article Title: Nitric oxide facilitates the S‐nitrosylation and deubiquitination of Notch1 protein to maintain cancer stem cells in human NSCLC

doi: 10.1111/jcmm.70203

Figure Lengend Snippet: NO inhibits the ubiquitination of Notch1. (A) mRNA level of Notch1 was measured in CSCs with or without L‐NAME (100 μM) treatment for 24 h. Mean ± SEM from four independent experiments. (B) Notch1 cleavage‐related mRNA expressions in non‐CSCs and CSCs was determined with qPCR. Mean ± SEM from six independent experiments. (C) CSCs were treated with or without L‐NAME (100 μM) for 24 h and detected for Notch1 cleavage‐related mRNA expressions by qPCR. Mean ± SEM from 6 to 8 independent experiments. (D) CSCs were treated with L‐NAME (100 μM) in the presence or absence of the MG132 (10 μM) for 4 h. Protein expression of Notch1 was detected by immunoblot. Mean ± SEM from four independent experiments. (E) Notch1 ubiquitination in non‐CSCs and CSCs was analysed using endogenous immunoprecipitated Notch1 protein. * p < 0.05, ** p < 0.01 with paired t ‐test (A, C), unpaired t ‐test (B) and ANOVA plus Turkey's method (D).

Article Snippet: Human Notch1 intracellular domain plasmid (#130934) was from Addgene.

Techniques: Ubiquitin Proteomics, Expressing, Western Blot, Immunoprecipitation

Journal: Journal of Cellular and Molecular Medicine

Article Title: Nitric oxide facilitates the S‐nitrosylation and deubiquitination of Notch1 protein to maintain cancer stem cells in human NSCLC

doi: 10.1111/jcmm.70203

Figure Lengend Snippet: NO inhibits the ubiquitination of Notch1 by facilitating its interaction with UCHL1. (A) Potential DUBs and E3 ligase of Notch1 obtained from the UbiBrowser database. (B, C) DUBs and E3 ligase mRNA expressions in non‐CSCs and CSCs were determined with qPCR. Mean ± SEM from six independent experiments. (D) Immunoblot analysis of UCHL1 protein in non‐CSCs and CSCs. Mean ± SEM from five independent experiments. (E) Co‐immunoprecipitation analysis of Notch1‐UCHL1 interaction in non‐CSCs and CSCs. (F) Genetic knockdown efficiency of UCHL1 in CSCs by lentiviral shRNA transfections. Mean ± SEM from four independent experiments. (G) Immunoblot analysis of Notch1 protein in CSCs transfected with or without the independent UCHL1 shRNAs. Mean ± SEM from four independent experiments. (H) Genetic knockdown efficiency of WWP1, MDM2, USP9X and PSMD7 in CSCs by lentiviral shRNA transfections. Mean ± SEM from 4 to 6 independent experiments. (I) Immunoblot analysis of Notch1 protein in CSCs transfected with or without the independent WWP1, MDM2, USP9X and PSMD7 shRNAs. (J) Immunoprecipitation analysis of ubiquitination of Notch1 in CSCs that were transfected with shUCHL1. (K) CSCs were transfected with UCHL1 shRNAs in the presence or absence of the DETA NONOate (20 μM). The protein of Notch1 was analysed with immunoblot. Mean ± SEM from four independent experiments. (L) Immunoprecipitation analysis of Notch1 ubiquitination in CSCs that were transfected with UCHL1 shRNAs together with DETA NONOate (20 μM). (M) CSCs transfected with UCHL1 shRNAs were analysed for expression of stem‐related transcripts. Mean ± SEM from 5 to 6 independent experiments. * p < 0.05, ** p < 0.01, *** p < 0.001 and **** p < 0.0001 with paired t ‐test (M) and ANOVA plus Turkey's method (F–H, K).

Article Snippet: Human Notch1 intracellular domain plasmid (#130934) was from Addgene.

Techniques: Ubiquitin Proteomics, Western Blot, Immunoprecipitation, Knockdown, shRNA, Transfection, Expressing

Journal: Journal of Cellular and Molecular Medicine

Article Title: Nitric oxide facilitates the S‐nitrosylation and deubiquitination of Notch1 protein to maintain cancer stem cells in human NSCLC

doi: 10.1111/jcmm.70203

Figure Lengend Snippet: NO‐facilitated s‐nitrosylation of Notch1 promotes its binding to UCHL1. (A) Biotin switch assay was employed to detect the SNO‐Notch1 levels in both non‐CSCs and CSCs. (B) The SNO‐Notch1 level in DETA NONOate‐treated (20 μM, 24 h) CSCs were detected by biotin switch assay. (C) The SNO‐Notch1 level in L‐NAME‐treated (100 μM, 24 h) CSCs were detected by biotin switch assay. (D) Notch1 protein levels in CSCs with or without ODQ (10 μM, 24 h) were analysed by immunoblotting. Mean ± SEM from four independent experiments. (E) Immunoprecipitation analysis of Notch1 ubiquitination in CSCs in the presence or absence of L‐NAME (100 μM) for 24 h. (F) Immunoprecipitation analysis of Notch1 ubiquitination in CSCs treated with DETA NONOate (20 μM) for 24 h. (G) Co‐immunoprecipitation analysis of Notch1 and UCHL1 interaction in CSCs in the presence or absence of DETA NONOate (20 μM). (H) Co‐immunoprecipitation analysis of Notch1 and UCHL1 interaction in CSCs with or without L‐NAME (100 μM).

Article Snippet: Human Notch1 intracellular domain plasmid (#130934) was from Addgene.

Techniques: Binding Assay, Biotin Switch Assay, Western Blot, Immunoprecipitation, Ubiquitin Proteomics

Journal: Journal of Cellular and Molecular Medicine

Article Title: Nitric oxide facilitates the S‐nitrosylation and deubiquitination of Notch1 protein to maintain cancer stem cells in human NSCLC

doi: 10.1111/jcmm.70203

Figure Lengend Snippet: Targeting UCHL1 and NO downregulates the expression levels of Notch1 and CD133. (A) Representative of PDO‐primary tumour pair with immunostaining for CD31. Nuclei were stained with Hoechst. Scale bar, 20 μm. (B) Representative of PDO‐primary tumour pair with immunostaining for PanCK. Nuclei were stained with Hoechst. Scale bar, 20 μm. (C) Representative HE staining of PDO‐primary tumour pairs. Scale bars, 100 μm. (D, E) PDOs treated with or without DETA NONOate (20 μM) were analysed for CD133 and Notch1 protein. Representatives from six independent experiments. (F, G) PDOs were transfected with UCHL1 shRNAs in the presence or absence of the DETA NONOate (20 μM), followed by analysis of CD133 and Notch1 protein. Representatives from 4 to 5 independent experiments. (H, I) PDOs exposed to 10 Gy x‐rays were treated with L‐NAME (100 μM), followed by analysis of CD133 and Notch1 protein. Representatives from 3 to 5 independent experiments. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001 with paired t ‐test (D, E) and ANOVA plus Turkey's method (F–I).

Article Snippet: Human Notch1 intracellular domain plasmid (#130934) was from Addgene.

Techniques: Expressing, Immunostaining, Staining, Transfection

Journal: Breast Cancer Research : BCR

Article Title: Oestrogen increases the activity of oestrogen receptor negative breast cancer stem cells through paracrine EGFR and Notch signalling

doi: 10.1186/bcr3396

Figure Lengend Snippet: Systemic oestrogen signalling is mediated by EGFR and Notch . ( A ) Representative Western blot showing expression of cleaved (active) Notch1 (N1-ICD) following culture ± 1 nM 17β-estradiol ± 10 μM GSI. ( Bi ) Representative Western blot showing expression of Notch ligands in sorted MCF7 cells (left) and, where available, metastatic cells (right). ( Bii ) Densitometric analysis of three independent repeats of MCF7 sorting and of a single experiment for primary cells. Comparisons between population 1 (CSC enriched) and other populations are displayed. ( C and D ) Mammosphere formation was assessed following culture with 1 nM 17β-estradiol ± gamma secretase inhibitor (GSI) alone and in combination with gefitinib. Fold change is normalised to control, untreated cells represented as line. ( E ) Representative image of protein levels of ERK and phosphorylated (actived) ERK following culture for 48 hours in monolayer ± 10 μM GSI. Means plotted ± SEM, * P < 0.05, ** P < 0.01, *** P < 0.001 compared to E2 treated. # P < 0.05 compared to control cells.

Article Snippet: Primary antibodies included: SP1-ER (RM-9101-SO, Thermo Fisher Scientific, Basingstoke, UK), Cleaved N1-ICD (100-401-407, Rockland, Gilbertsville, USA), Delta1 (Santa Cruz Biotechnology, Santa Cruz, CA, USA, sc-12530), Delta4 (Abcam, Cambridge, UK, ab7280), Jagged1 (Santa Cruz Biotechnology, sc-6011), Jagged2 (Santa Cruz Biotechnology, sc-08157), Actin (Santa Cruz Biotechnology, sc-1616), ERK (Abcam, ab2430) and ERK phospho-Y992 (Abcam, ab81440).

Techniques: Western Blot, Expressing, Control

Journal: CytoJournal

Article Title: Roxadustat: A catalyst for diabetic wound healing through re-epithelialization and angiogenesis

doi: 10.25259/Cytojournal_235_2024

Figure Lengend Snippet: Roxadustat (FG-4592) promotes dedifferentiation of keratinocytes and angiogenesis in diabetic mice. (a) Expression levels of integrin β1, K14, K10, K1, and Notch1 NICD evaluated by Western blot in the middle and at the end of wound healing. (b-f) Corresponding quantitative analysis. (g) CD31 and VEGF expression levels evaluated by Western blot. (h and i) Corresponding quantitative analysis. n = 3 in each group. ✶ P < 0.05. NICD: Notch Intracellular Domain, VEGF: Vascular endothelial growth factor, K14: Keratin 14, K10: Keratin 10, K1: Keratin 1.

Article Snippet: The primary antibodies for HIF-1α (1:100, 66730-1-Ig, Proteintech, China) and Notch1 ICD (1:100, PAB35376 , Bioswamp, China) were incubated overnight at 4°C.

Techniques: Expressing, Western Blot

Journal: CytoJournal

Article Title: Roxadustat: A catalyst for diabetic wound healing through re-epithelialization and angiogenesis

doi: 10.25259/Cytojournal_235_2024

Figure Lengend Snippet: Roxadustat (FG-4592) promotes dedifferentiation through interaction between HIF-1α and NICD. (a) Immunofluorescence showing the expression and co-localization of HIF-1α and NICD in HaCaT cells under different conditions; bar = 100 μm. (b) Western blot showing the expression levels of HIF-1α, NICD, K14, and integrin-β1 in HaCaT cells under different conditions. (c-f) Quantitative analysis of WB. n = 3 in each group; ✶ P < 0.05. (g) Co-IP confirming the interaction between HIF-1α and NICD. HIF-1: Hypoxia-inducible factor 1, NICD: Notch intracellular domain, WB: Western blot, Co-IP: Co-immunoprecipitation, K14: Keratin 14.

Article Snippet: The primary antibodies for HIF-1α (1:100, 66730-1-Ig, Proteintech, China) and Notch1 ICD (1:100, PAB35376 , Bioswamp, China) were incubated overnight at 4°C.

Techniques: Immunofluorescence, Expressing, Western Blot, Co-Immunoprecipitation Assay, Immunoprecipitation

Journal: CytoJournal

Article Title: Roxadustat: A catalyst for diabetic wound healing through re-epithelialization and angiogenesis

doi: 10.25259/Cytojournal_235_2024

Figure Lengend Snippet: Schematic model of roxadustat reversing the “high differentiated and low proliferation” state of keratinocytes in diabetic wounds. In normal skin, keratinocytes maintain a relatively stable rhythm of proliferation and differentiation. Injury can activate the process of wound repair, upregulate HIF-1 signal, downregulate Notch1 signal, and make keratinocytes in a state of “high proliferation and low differentiation.” In the context of diabetes, the skin is usually thin and wound healing is delayed, HIF-1 signaling is inhibited, and Notch1 signaling is continuously activated, making keratinocytes in a state of “low proliferation and high differentiation.” FG-4592 upregulates the inhibited HIF-1 signaling and downregulates the hyperactivated Notch1 signaling, which both benefit diabetic wound re-epithelialization. By Fig draw (version 2.0, www.figdraw.com ). HIF-1: Hypoxia-inducible factor 1.

Article Snippet: The primary antibodies for HIF-1α (1:100, 66730-1-Ig, Proteintech, China) and Notch1 ICD (1:100, PAB35376 , Bioswamp, China) were incubated overnight at 4°C.

Techniques: